**1. Introduction**

The flagellate protozoan *Trypanosoma cruzi* is the etiologic agent of Chagas disease or American trypanosomiasis, which affects 6–7 million people mainly in Latin America, with an increasing number of cases in non-endemic countries such as Canada, the United States of America, and some European countries [1]. When compared with other members of the genus Trypanosoma, the *T. cruzi* genome was expanded, being 2.3-fold larger than that of *T. brucei* and *T. rangeli*. Repetitive DNA sequences comprise about 52% of the *T. cruzi* genome [2–4]. The dramatic expansion and diversification of repetitive sequences, particularly of multigene family encoding proteins, such as surface proteins (TS (Trans-Sialidase), MASP (Mucin-Associated Surface Protein), mucins, gp63, Retrotransposon Hot Spot (RHS), and DGF-1 (Dispersed Gene Family-1)) may have contributed to the speciation of the *T. cruzi* taxon [2,5]. RHS proteins are coded by a multigene family found in the genus Trypanosoma.

RHS refers to a hot spot for retrotransposon insertion within the RHS gene. When retrotransposons are inserted in this site, they generate RHS pseudogenes carrying one or more retroelements flanked by two separate halves of RHS [6]. Multiple RHS genes have been annotated in the genomes of mammalian trypanosomes (African trypanosomes—*T. brucei*, *T. congolense*, and *T. vivax*; American trypanosomes—*T. cruzi*, *T. cruzi marinkellei*, and *T. rangeli*; and cosmopolitan trypanosomes—*T. theileri*, *T. evansi*, *T. conorhini*) and *T. grayi* isolated from reptiles.

RHS proteins were first identified in *T. brucei* and were classified into six subfamilies (RHS1 to RHS6) based on the C-terminal region sequence [6]. The RHS proteins of *T. brucei* share a highly conserved amino-terminal (N-terminal) region, while the carboxy-terminal (C-terminal) portion is highly variable [6]. The N-terminal region has an ATP/GTP binding motif encoded by five codons located upstream of the hot spot insertion site for the retrotransposons Ingi (an autonomous long interspersed element—LINE) and RIME (a non-autonomous short interspersed element—SINE). The pseudogene may be the result of homologous recombination between two RHS variants by crossing-over involving the 5′ region upstream of the retroelement insertion site. Retrotransposon insertion generates nonsense mutations or frameshifts within the coding sequence, resulting in truncated RHS proteins [6].

The role of the RHS family has been investigated in *T. brucei*, and it has been suggested that RHSs are involved in the control of the expansion of the retroelements in this organism [6,7]. Earlier studies in *T. brucei* showed an increase in the level of RHS transcripts after the ablation of argonaute protein, suggesting that the RHS family may be under the control of siRNA (small interfering RNA) [8]. High throughput analysis of small non-coding RNAs showed that a large number of pseudogene-derived siRNAs originated from pseudogene–pseudogene pairs, in which the major components were RHS pseudogenes [9], and it has been hypothesized that RHS pseudogenes in *T. brucei* are a source of antisense siRNAs, which regulate the expression of the RHS family. More recent studies proposed that the RHS family could be involved in the chromatin modeling [10], transcription elongation, and mRNA export in *T. brucei* [11].

Beyond an initial genomic analysis showing multiple RHS (gene) pseudogenes, little is known about the organization, structure, and expression of these genes and their products in *T. cruzi.* In the current study, we aimed to investigate the structure, evolution, and expression of the RHS multigene family in *T. cruzi*. We also provide insights into the strategies used by *T. cruzi* for preserving complete and functional RHS genes.
